Abstract Bronchopulmonary dysplasia (BPD) is one of the most common and important sequelae of premature birth, affecting as many as 30% of infants with birth weights less than 1500 g. BPD rates have been increasing among premature infants with gestational ages less than 28 weeks due to increasing survival of extremely low birth weight infants. Infants with severe BPD often require respiratory support after discharge from the hospital and suffer from consequences of chronic respiratory morbidity throughout their lives. They are also at higher risk of neurodevelopmental problems and death. Currently, there is a shortage of evidence-based safe treatments for BPD. Autophagy is a crucial catabolic pathway for cellular homeostasis. During autophagy, cytosolic substrates or impaired organelles are enclosed in autophagosomes, and transferred to lysosomes for digestion by cathepsins and other acid hydrolases. Autophagy is induced as an important part of the mammalian stress response and is believed to represent a cytoprotective response under most circumstances, but excessive or aberrant activation of autophagy can also lead to cell death. While molecular mechanisms that regulate the formation of autophagosome are well studied, the regulation of late digestive steps of autophagy remain relatively uncharacterized. Emerging data suggest an essential role for lysosomal cysteine cathepsins in regulation and execution of autophagy. Autophagy can be activated by hyperoxia exposure of the lung, which is an important factor in the pathogenesis of BPD. However, the potential role of autophagy in BPD remains to be elucidated. Our studies have demonstrated increased activity of lysosomal cysteine cathepsins in murine and baboon lungs with BPD. The goal of this proposal is to test the hypothesis that autophagic flux induces lysosomal cysteine cathepsin activation and plays a maladaptive role in neonatal hyperoxia-induced lung injury (nHILI). The proposed studies will examine the role of autophagic flux in lysosomal cysteine cathepsin activation and determine whether autophagy-deficient mice are protected from nHILI. They will also explore whether autophagic flux can be monitored with a fluorescent-labeled cathepsin activity based probe in a murine model of nHILI. Overall, these studies should provide important insights into the role of autophagy and cysteine cathepsin activation in nHILI, which can be exploited in future studies to identify new therapeutic targets for BPD. They also have the potential to identify a novel tool for dynamic monitoring of autophagic flux in nHILI.